Neisseria gonorrhoeae is a human pathogen that causes the sexually transmitted infection termed gonorrhea. The yearly incidence of gonorrhea worldwide (>100 million cases) is a major public health problem, which is compounded by the emergence of strains resistant to multiple antibiotics. The problem of antibiotic resistance expressed by certain clinical isolates has been made more worrisome recently with reports of strains expressing decreased susceptibility or resistance to the third generation cephalosporins (ceftriaxone and cefixime) that have been used for empiric therapy of gonorrhea in the United States. Symptomatic gonorrhea is largely due to the host's inflammatory response, which can result in severe complications for men and women. Invasive gonorrhea in the female patient can be particularly acute resulting in severe gynecologic consequences. This Merit Award renewal is dedicated to understanding the genetics and molecular control of an important modification of the gonococcal outer membrane that contributes to the ability of gonococci to resist certain mediators (complement and cationic antimicrobial peptides [CAMPs]) of innate host defense. This modification consists of the attachment of phosphoethanolamine (PEA) to the lipid A of lipooligosaccharide by an enzyme termed LptA. During the past funding period we showed that null mutations in the gene (lptA) encoding LptA rendered gonococci hyper-susceptible to complement and CAMPs, depressed the pro- inflammatory response induced by gonococci and decreased bacterial fitness during an experimental infection of the lower genital tract of female mice. Further, we uncovered a multi-layered, integrated and complex regulatory system that modulates lptA expression and, as a consequence, PEA decoration of lipid A, gonococcal susceptibility to CAMPs, in vivo fitness of gonococci and inflammation. This system is the focus of this renewal application. The overall regulatory system consists of multiple promoters within an operon for lptA expression, a phase variable polynucleotide repeat sequence within the lptA coding sequence that can cause truncation of LptA at high frequencies and the gene-activating capacity of a DNA-binding protein termed MisR. During the requested funding period, these regulatory processes will be defined and their impact on CAMP resistance will be determined. The studies embodied in Specific Aim 1 will define and characterize the promoters used for lptA expression, their differential use and response to sub-lethal levels of antimicrobials. Transcriptional factors that modulate use of these promoters will be identified so as to understand how lptA expression is controlled at the transcriptional level. Additionally, the phase variable production of LptA will be defined in laboratory-maintained and recent clinical strains. The studies embodied in Specific Aim 2 are dedicated to understanding the mechanisms by which MisR regulates lptA expression and how this control system influences gonococcal resistance to CAMPs and production of unique lipid A structures. The successful completion of the proposed work will significantly advance our knowledge regarding how gonococci can evade host defenses and modulate pro-inflammatory responses through the use of gene control systems. Importantly, the results should also provide important insights for the future development of novel antimicrobial agents and therapeutic strategies that can be used in the treatment of gonorrhea.